Cloning And Expression Of Serpin From Bifidobacteria And Its Preliminary Function Study

Bifidobacteria is autochthonous and predominant species in human intestine, which plays an important role in maintaining the microecology balance and improving the host health by adhering and colonizing intestinal epithelial cells and thus forming biological barriers together with other anaerobe against pathogen invasion. Adherence and immunity were the major basis for the physiological function of Bifidobacterium sp. However, the mechanisms of Bifidobacterium adhering to the intestinal epithelial and its relevant substances involving into modulation of host immunity was not totally clear. Currently studies on the adhesion and immunity of probiotics were mainly focused on out-membrane proteins of cells. As one of the outer membrane protein of Bifidobacterium, Serpin (serine protease inhibitor) attracted few researching in the last three years.Serpins were a superfamily of proteins (350-500 amino acids in size) of serine protease inhibitor. By regulating the activity of serine proteases and homocysteine enzyme, Serpins played a great role in higher eukaryote organisms, such as coagulation, fibrinolysis, immune response, cell differentiation and apoptosis. However, the function of Serpins in prokaryote was still less studied. In 2006 the Swiss Nestle Research Center found that Serpin from B. longum was efficient to inhibit the activities of exogenous proteinase in the gastrointestinal tract, which provided B. longum a competitive advantage to adhere in the intestinal tract. To clarify the distribution of Serpin in species of Bifidobacteria,18 strains was investigated, and thereof 3 strains, namely, B. infantis WBAN07, B. bifidum WBBI02 and B. longum NCC2705 were proved to own serpin gene by PCR and Southern-blotting, their sequence homology was 99.9%.. Sequencing and bioinformation analysis disclosed that eight nucleotide variations took place, amino acid alignment revealed that two amino acid changes (149th His and 770th Gln in B. longum NCC2705,149th Arg in B. bifidum WBBI02, and 149th His and 770th Gln in B. infantis WBAN07) corresponding to the nucleotide variations occurred in the three accessions.Considering the strong adherence of B. bifidum WBBI02, we focused on the expression and functional study of its serpin. Therein, the hydrophobic area in two ends of serpin was cut and a recombinant serpin gene expression system of B. bifidum WBBI02 was ligated into pBX2 vector in frame with the N-terminal 6×His tag. pBX2-WBBI02 was transformed into Escherichia coli (BL21 strain), and the expression of serpin was carried out by inducing with 0.2 mmol/L IPTG at 25℃for 3-4 h. A 32 kD serpin protein was purified with the aid of.NTA His-Bind resin column. Preliminary test of the biological activities of serpin showed that serpin was efficient to inhibit eukaryotic a-chymotrypsin and pancreatic elastase by 90% and 97%, respectively, and further enhance the adherence of Bifidobacteria to HT-29 cells confirmed by microscopal observation.In summary, the gene cloning and expression of Serpin from B. bifidum WBBI02, and its functional study provided a solid basis for broad investigation on the serpin from other probiotics and offer a theoretical and technological method for guiding the future research on function i.e. adherence and immunity of surface proteins from probiotics.